KARP-1 is induced by DNA damage in a p53- and ataxia telangiectasia mutated-dependent fashion

The KARP-1 (Ku86 Autoantigen Related Protein-1) gene, which is expressed from the human Ku86 autoantigen locus, appears to play a role in mammalian DNA double-strand break repair as a regulator of the DNA-dependent protein kinase complex. Here we demonstrate that KARP-1 gene expression is significantly up-regulated following exposure of cells to DNA damage. KARP-1 mRNA induction was completely dependent on the ataxia telangiectasia and p53 gene products, consistent with the presence of a p53 binding site within the second intron of the KARP-1 locus. These observations link ataxia telangiectasia, p53, and KARP-1 in a common pathway.

Activities and response to DNA damage of latent and active sequence-specific DNA binding forms of mouse p53

The mouse p53 protein generated by alternative splicing (p53as) has amino acid substitutions at its C terminus that result in constitutively active sequence-specific DNA binding (active form), whereas p53 protein itself binds inefficiently (latent form) unless activated by C-terminal modification. Exogenous p53as expression activated transcription of reporter plasmids containing p53 binding sequences and inhibited growth of mouse and human cells lacking functional endogenous p53. Inducible p53as in stably transfected p53 null fibroblasts increased p21WAF1/Cip-1/Sdi and decreased bcl-2 protein steady-state levels. Endogenous p53as and p53 proteins differed in response to cellular DNA damage. p53 protein was induced transiently in normal keratinocytes and fibroblasts whereas p53as protein accumulation was sustained in parallel with induction of p21WAF1/Cip-1/Sdi protein and mRNA, in support of p53as transcriptional activity. Endogenous p53 and p53as proteins in epidermal tumor cells responded to DNA damage with different kinetics of nuclear accumulation and efficiencies of binding to a p53 consensus DNA sequence. A model is proposed in which C-terminally distinct p53 protein forms specialize in functions, with latent p53 forms primarily for rapid non-sequence-specific binding to sites of DNA damage and active p53 forms for sustained regulation of transcription and growth.

Identification of the prooxidant site of human ceruloplasmin: A model for oxidative damage by copper bound to protein surfaces

Free transition metal ions oxidize lipids and lipoproteins in vitro; however, recent evidence suggests that free metal ion-independent mechanisms are more likely in vivo. We have shown previously that human ceruloplasmin (Cp), a serum protein containing seven Cu atoms, induces low density lipoprotein oxidation in vitro and that the activity depends on the presence of a single, chelatable Cu atom. We here use biochemical and molecular approaches to determine the site responsible for Cp prooxidant activity. Experiments with the His-specific reagent diethylpyrocarbonate (DEPC) showed that one or more His residues was specifically required. Quantitative [14C]DEPC binding studies indicated the importance of a single His residue because only one was exposed upon removal of the prooxidant Cu. Plasmin digestion of [14C]DEPC-treated Cp (and N-terminal sequence analysis of the fragments) showed that the critical His was in a 17-kDa region containing four His residues in the second major sequence homology domain of Cp. A full length human Cp cDNA was modified by site-directed mutagenesis to give His-to-Ala substitutions at each of the four positions and was transfected into COS-7 cells, and low density lipoprotein oxidation was measured. The prooxidant site was localized to a region containing His426 because CpH426A almost completely lacked prooxidant activity whereas the other mutants expressed normal activity. These observations support the hypothesis that Cu bound at specific sites on protein surfaces can cause oxidative damage to macromolecules in their environment. Cp may serve as a model protein for understanding mechanisms of oxidant damage by copper-containing (or -binding) proteins such as Cu, Zn superoxide dismutase, and amyloid precursor protein.

Collagenase-3 Induction in Rat Lung Fibroblasts Requires the Combined Effects of Tumor Necrosis Factor-α and 12-Lipoxygenase Metabolites: A Model of Macrophage-induced, Fibroblast-driven Extracellular Matrix Remodeling during Inflammatory Lung Injury

The mechanisms responsible for the induction of matrix-degrading proteases during lung injury are ill defined. Macrophage-derived mediators are believed to play a role in regulating synthesis and turnover of extracellular matrix at sites of inflammation. We find a localized increase in the expression of the rat interstitial collagenase (MMP-13; collagenase-3) gene from fibroblastic cells directly adjacent to macrophages within silicotic rat lung granulomas. Conditioned medium from macrophages isolated from silicotic rat lungs was found to induce rat lung fibroblast interstitial collagenase gene expression. Conditioned medium from primary rat lung macrophages or J774 monocytic cells activated by particulates in vitro also induced interstitial collagenase gene expression. Tumor necrosis factor-α (TNF-α) alone did not induce interstitial collagenase expression in rat lung fibroblasts but did in rat skin fibroblasts, revealing tissue specificity in the regulation of this gene. The activity of the conditioned medium was found to be dependent on the combined effects of TNF-α and 12-lipoxygenase-derived arachidonic acid metabolites. The fibroblast response to this conditioned medium was dependent on de novo protein synthesis and involved the induction of nuclear activator protein-1 activity. These data reveal a novel requirement for macrophage-derived 12-lipoxygenase metabolites in lung fibroblast MMP induction and provide a mechanism for the induction of resident cell MMP gene expression during inflammatory lung processes.

Endogenous tumor necrosis factor protects the adult cardiac myocyte against ischemic-induced apoptosis in a murine model of acute myocardial infarction

Previous studies have shown that proinflammatory cytokines, such as tumor necrosis factor (TNF), are expressed after acute hemodynamic overloading and myocardial ischemia/infarction. To define the role of TNF in the setting of ischemia/infarction, we performed a series of acute coronary artery occlusions in mice lacking one or both TNF receptors. Left ventricular infarct size was assessed at 24 h after acute coronary occlusion by triphenyltetrazolium chloride (TTC) staining in wild-type (both TNF receptors present) and mice lacking either the type 1 (TNFR1), type 2 (TNFR2), or both TNF receptors (TNFR1/TNFR2). Left ventricular infarct size as assessed by TTC staining was significantly greater (P < 0.005) in the TNFR1/TNFR2-deficient mice (77.2% ± 15.3%) when compared with either wild-type mice (46.8% ± 19.4%) or TNFR1-deficient (47.9% ± 10.6%) or TNFR2-deficient (41.6% ± 16.5%) mice. Examination of the extent of necrosis in wild-type and TNFR1/TNFR2-deficient mice by anti-myosin Ab staining demonstrated no significant difference between groups; however, the peak frequency and extent of apoptosis were accelerated in the TNFR1/TNFR2-deficient mice when compared with the wild-type mice. The increase in apoptosis in the TNFR1/TNFR2-deficient mice did not appear to be secondary to a selective up-regulation of the Fas ligand/receptor system in these mice. These data suggest that TNF signaling gives rise to one or more cytoprotective signals that prevent and/or delay the development of cardiac myocyte apoptosis after acute ischemic injury.

Ultraviolet radiation, but not γ radiation or etoposide-induced DNA damage, results in the phosphorylation of the murine p53 protein at serine-389

Polyclonal antibodies were produced and purified that selectively react with a p53 epitope containing the murine phosphoserine-389 or the human phosphoserine-392 residue, but not the unphosphorylated epitope. These antibodies, termed alpha-392, were employed to demonstrate that the phosphorylation of this serine-389 residue in the p53 protein occurs in vivo in response to ultraviolet radiation of cells containing the p53 protein. After ultraviolet radiation of cells in culture, p53 levels increase and concomitantly serine-389 is phosphorylated in these cells. By contrast, the serine-389 phosphorylation of the p53 protein was not detected by these antibodies in the increased levels of p53 protein made in response to γ radiation or the treatment of cells with etoposide. These results demonstrate an ultraviolet responsive and specific phosphorylation site at serine-389 of the mouse or serine-392 of the human p53 protein. Previous studies have demonstrated that this phosphorylation of p53 activates the protein for specific DNA binding. This study demonstrates in vivo a unique phosphorylation site in the p53 protein that responds to a specific type of DNA damage.

Corepressor-induced organization and assembly of the biotin repressor: A model for allosteric activation of a transcriptional regulator

The Escherichia coli biotin repressor binds to the biotin operator to repress transcription of the biotin biosynthetic operon. In this work, a structure determined by x-ray crystallography of a complex of the repressor bound to biotin, which also functions as an activator of DNA binding by the biotin repressor (BirA), is described. In contrast to the monomeric aporepressor, the complex is dimeric with an interface composed in part of an extended β-sheet. Model building, coupled with biochemical data, suggests that this is the dimeric form of BirA that binds DNA. Segments of three surface loops that are disordered in the aporepressor structure are located in the interface region of the dimer and exhibit greater order than was observed in the aporepressor structure. The results suggest that the corepressor of BirA causes a disorder-to-order transition that is a prerequisite to repressor dimerization and DNA binding.

Segregation of DNA polynucleotide strands into sister chromatids and the use of endoreduplicated cells to track sister chromatid exchanges induced by crosslinks, alkylations, or x-ray damage.

The method of Matsumoto and Ohta [Matsumoto, K. & Ohta, T. (1992) Chromosoma 102, 60-65; Matsumoto, K. & Ohta, T. (1995) Mutat. Res. 326, 93-98] to induce large numbers of endoreduplicated Chinese hamster ovary cells has now been coupled with the fluorescence-plus-Giemsa method of Perry and Wolff [Perry, P. & Wolff, S. (1974) Nature (London) 251, 156-158] to produce harlequin endoreduplicated chromosomes that after the third round of DNA replication are composed of a chromosome with a light chromatid and a dark chromatid in close apposition to its sister chromosome containing two light chromatids. Unless the pattern is disrupted by sister chromatid exchange (SCE), the dark chromatid is always in the center, so that the order of the chromatids is light-dark light-light. The advent of this method, which permits the observation of SCEs in endoreduplicated cells, makes it possible to determine with great ease in which cell cycle an SCE occurred. This now allows us to approach several vexing questions about the induction of SCEs (genetic damage and its repair) after exposure to various types of mutagenic carcinogens. The present experiments have allowed us to observe how many cell cycles various types of lesions that are induced in DNA by a crosslinking agent, an alkylating agent, or ionizing radiation, and that are responsible for the induction of SCEs, persist before being repaired and thus lose their ability to inflict genetic damage. Other experiments with various types of mutagenic carcinogens and various types of cell lines that have defects in different DNA repair processes, such as mismatch repair, excision repair, crosslink repair, and DNA-strand-break repair, can now be carried out to determine the role of these types of repair in removing specific types of lesions.

Role of neuronal nitric oxide in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurotoxicity.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes nigrostriatal dopaminergic pathway damage similar to that observed in Parkinson disease (PD). To study the role of NO radical in MPTP-induced neurotoxicity, we injected MPTP into mice in which nitric oxide synthase (NOS) was inhibited by 7-nitroindazole (7-NI) in a time- and dose-dependent fashion. 7-NI dramatically protected MPTP-injected mice against indices of severe injury to the nigrostriatal dopaminergic pathway, including reduction in striatal dopamine contents, decreases in numbers of nigral tyrosine hydroxylase-positive neurons, and numerous silver-stained degenerating nigral neurons. The resistance of 7-NI-injected mice to MPTP is not due to alterations in striatal pharmacokinetics or content of 1-methyl-4-phenylpyridinium ion (MPP+), the active metabolite of MPTP. To study specifically the role of neuronal NOS (nNOS), MPTP was administered to mutant mice lacking the nNOS gene. Mutant mice are significantly more resistant to MPTP-induced neurotoxicity compared with wild-type littermates. These results indicate that neuronally derived NO mediates, in part, MPTP-induced neurotoxicity. The similarity between the MPTP model and PD raises the possibility that NO may play a significant role in the etiology of PD.

Elevated interleukin 6 is induced by prostaglandin E2 in a murine model of inflammation: possible role of cyclooxygenase-2.

Injection of mineral oils such as pristane into the peritoneal cavities of BALB/c mice results in a chronic peritonitis associated with high tissue levels of interleukin 6 (IL-6). Here we show that increased prostaglandin E2 (PGE2) synthesis causes induction of IL-6 and that expression of an inducible cyclooxygenase, Cox-2, may mediate this process. Levels of both PGE2 and IL-6 are elevated in inflammatory exudates from pristane-treated mice compared with lavage samples from untreated mice. The Cox-2 gene is induced in the peritoneal macrophage fraction isolated from the mice. A cause and effect relationship between increased macrophage PGE2 and IL-6 production is shown in vitro. When peritoneal macrophages are activated with an inflammatory stimulus (polymerized albumin), the Cox-2 gene is induced and secretion of PGE2 and IL-6 increases, with elevated PGE2 appearing before IL-6. Cotreatment with 1 microM indomethacin inhibits PGE2 production by the cells and reduces the induction of IL-6 mRNA but has no effect on Cox-2 mRNA, consistent with the fact that the drug inhibits catalytic activity of the cyclooxygenase but does not affect expression of the gene. Addition of exogenous PGE2 to macrophages induces IL-6 protein and mRNA synthesis, indicating that the eicosanoid stimulates IL-6 production at the level of gene expression. PGE2-stimulated IL-6 production is unaffected by addition of indomethacin. Taken together with the earlier finding that indomethacin diminishes the elevation of IL-6 in pristane-treated mice, the results show that PGE2 can induce IL-6 production in vivo and implicate expression of the Cox-2 gene in the regulation of this cytokine.

Progression of human breast cancers to the metastatic state is linked to hydroxyl radical-induced DNA damage.

Hydroxyl radical damage in metastatic tumor DNA was elucidated in women with breast cancer, and a comparison was made with nonmetastatic tumor DNA. The damage was identified by using statistical models of modified base and Fourier transform-infrared spectral data. The modified base models revealed a greater than 2-fold increase in hydroxyl radical damage in the metastatic tumor DNA compared with the nonmetastatic tumor DNA. The metastatic tumor DNA also exhibited substantially greater base diversity than the nonmetastatic DNA, and a progression of radical-induced base damage was found to be associated with the growth of metastatic tumors. A three-dimensional plot of principal components from factor analysis, derived from infrared spectral data, also showed that the metastatic tumor DNA was substantially more diverse than the tightly grouped nonmetastatic tumor DNA. These cohesive, independently derived findings suggest that the hydroxyl radical generates DNA phenotypes with various metastatic potentials that likely contribute to the diverse physiological properties and heterogeneity characteristic of metastatic cell populations.

Overexpression of a Rrp1 transgene reduces the somatic mutation and recombination frequency induced by oxidative DNA damage in Drosophila melanogaster.

Recombination repair protein 1 (Rrp1) includes a C-terminal region homologous to several DNA repair proteins, including Escherichia coli exonuclease III and human APE, that repair oxidative and alkylation damage to DNA. The nuclease activities of Rrp1 include apurinic/apyrimidinic endonuclease, 3'-phosphodiesterase, 3'-phosphatase, and 3'-exonuclease. As shown previously, the C-terminal nuclease region of Rrp1 is sufficient to repair oxidative- and alkylation-induced DNA damage in repair-deficient E. coli mutants. DNA strand-transfer and single-stranded DNA renaturation activities are associated with the unique N-terminal region of Rrp1, which suggests possible additional functions that include recombinational repair or homologous recombination. By using the Drosophila w/w+ mosaic eye system, which detects loss of heterozygosity as changes in eye pigmentation, somatic mutation and recombination frequencies were determined in transgenic flies overexpressing wild-type Rrp1 protein from a heat-shock-inducible transgene. A large decrease in mosaic clone frequency is observed when Rrp1 overexpression precedes treatment with gamma-rays, bleomycin, or paraquat. In contrast, Rrp1 overexpression does not alter the spot frequency after treatment with the alkylating agents methyl methanesulfonate or methyl nitrosourea. A reduction in mosaic clone frequency depends on the expression of the Rrp1 transgene and on the nature of the induced DNA damage. These data suggest a lesion-specific involvement of Rrp1 in the repair of oxidative DNA damage.